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Examination of Serum miRNA Levels in Kidney Transplant Recipients With Acute Rejection

2014; Wolters Kluwer; Volume: 97; Issue: 4 Linguagem: Inglês

10.1097/01.tp.0000441098.68212.de

1534-6080

Gareth J. Betts, Sushma Shankar, Sam Sherston, Peter J. Friend, Kathryn J. Wood,

Circular RNAs in diseases

MiRNAs inhibit translation of messenger RNAs and are highly stable in serum (1, 2) and urine (3), making them potential biomarkers of pathology, including rejection of solid organ allografts (4–8). miR-10a, -10b, 30c, 32, 142-3p, -223, and let-7c (4, 9) are differentially expressed in allograft biopsies undergoing acute rejection (AR) in kidney transplant recipients (KTRs) compared with healthy allograft biopsies, as is miR-483 compared with nontransplanted kidney (10). miR-30c, 32, -142-3p, -204, -211, -223, -483, and -663b are differentially expressed in KTR biopsies with interstitial fibrosis/tubular atrophy (IF/TA) compared with healthy allografts (11, 12). miR 142-3p, -204, and -211 in KTRs with IF/TA (12) and miR-10a and -10b in KTRs with acute rejection (3) was differentially expressed in urine compared with stable KTRs. Several miRNA are elevated in patient serum during acute rejection of liver (2) and heart allografts (13). Twelve KTRs receiving basiliximab, tacrolimus, and steroids, with azathioprine or mycophenolate mofetil, were enrolled (REC number 07/H0603/26). All patients were negative for donor-specific antibodies (DSA) pretransplantation and so did not receive pretransplant desensitization. All patients had primary renal function after transplant. Seven of these patients underwent a single biopsy proven acute cellular rejection (AR) within the first year posttransplantation and an additional patient at day 404 posttransplant. All biopsies were scored according to the Banff criteria: three biopsies scored 4 IA, one biopsy scored 4 IIA, one biopsy scored 4 IIB, and three biopsies scored 3. All biopsies were C4d negative. Each AR episode was successfully treated with three daily doses of 500-mg methylprednisolone, as measured by restored eGFR to baseline, posttreatment. The remaining four patients maintained healthy graft function throughout and were matched for HLA, race, induction and maintenance therapy, and initial graft function to patients with AR. There was no difference in PRA between control and AR groups. All 12 patients maintained healthy graft function and eGFR at baseline at 1 year posttransplantation. Within the AR group, miRNA levels were measured at several time points: PRE-AR, AR, POST-AR, and 1 year posttransplantation. MiRNA levels in the control group were measured at time points parallel to those of AR patients at time of AR. We hypothesized that miRNAs demonstrated to be differentially expressed in biopsy material, and urine of KTRs undergoing rejection is also differently expressed in serum; we therefore analyzed the expression of several candidate miRNAs (Fig. 1). mirVana PARIS kit was used to purify RNA from KTR serum archived at −40°C before RT, preamplification, and qPCR (Life Technologies).FIGURE 1: Comparison of miR-10a, miR-10b, miR-30c, miR-142-3p, miR-204, miR-483, miR-663, and Let-7c levels in the serum. Levels of miR-211 and miR-32 were too low in serum to measure accurately. miR-16 was used as the endogenous control. Pretransplant serum samples were collected from controls and AR patients. ΔCt values were calculated (Ct miRNA – Ct miR-16), before using the preoperative ΔCt as the calibrator (ΔΔCt=ΔCt time point - ΔCt preoperative time point) for all posttransplant samples and subsequently determining the 2-ΔΔCt. AR episode serum samples were collected at days 4, 6, 7, 41, 48, 82, 210, and 393 days posttransplant (1-year sample was not measured for day 393 AR patient). (PRE-AR: available for 3/8 AR patients, collected on average of 135 days after transplant); (POST-AR: available for 6/8 AR patients; on average, 174 days posttransplant); (1 yr: available for 6/8 AR patients). 3/8 AR episodes were histologically defined as borderline rejection. miRNA levels were not distinguishable between AR and borderline AR samples and are analyzed as one group throughout. Control and AR KTRs (black-filled circles) are distinguished from KTR with borderline rejection. Three patients with borderline AR are distinguished by blue squares (patient 1), green diamonds (patient 2), and red triangles (patient 3) at each time point. For 4 of 8 patients with AR, serum from a separate group of healthy KTRs was measured alongside. Bars represent mean data with SEM shown. Comparisons between samples obtained at different time points from the same patient and similar time points from different patients were made using Wilcoxon and Mann-Whitney statistical tests, respectively.There was a significant reduction of miR-223 and miR-10a levels (P<0.05) and a trend for reduced miR-30c, miR-142-3p, and miR-483 levels (P=0.063) in serum 1 year posttransplant compared with the AR samples, whereas Let-7c, miR-10b, miR-204, and miR-663 levels were not different (Fig. 1). Interestingly, there was no difference in the expression of any miRNA measured between AR and control samples (Fig. 1). AR episodes occurred soon after transplant, with 3/8 AR within a week and two more within 50 days of transplant. It is possible that the difference between AR and 1 year samples is due to a decrease in serum miRNA levels at a greater interval since transplant, independently of AR. The number of PRE-AR samples available was too low to provide conclusive results, but an elevated expression of miR-223 and miR-10a compared with 1-year samples, independent of AR, is indicated (Fig. 1). Analysis of AR and POST-AR samples (mean POST-AR =174 days posttreatment) suggests levels began to reduce closer to the date of the transplant, with a trend for reduced levels of miR-223 and miR-10a (P=0.063). A similar trend of reduced miR-204 level was observed POST-AR compared with AR (P=0.063). The miR-204 level was significantly higher in controls, measured shortly after transplantation, compared with 1 year after transplant in KTRs that had an AR episode (P<0.05), highlighting the possibility that miRNA expression levels observed 1 year posttransplant might fall independently of an AR episode. Examination of control patients early posttransplant and at 1 year posttransplant could elucidate whether reduced miR-223 and miR-10a levels with extended time posttransplant are associated with factors independent of AR, including longer exposure to posttransplant maintenance immunosuppression. These data have to be interpreted cautiously because of the small patient cohort. It seems that miRNA levels in kidney biopsies and urine previously described to be differentially expressed in healthy allografts and those undergoing rejection are not universally translated to differential expression in the serum. These data also indicate that miRNA levels alter after transplantation, independently of AR, highlighting the importance of carefully matching cohorts of KTRs with healthy allografts and AR. Gareth Betts Sushma Shankar Sam Sherston Peter Friend Kathryn J. Wood Transplantation Research Immunology Group Nuffield Department of Surgical Sciences University of Oxford John Radcliffe Hospital Oxford, UK ACKNOWLEDGMENT The authors thank Sally Ruse for phlebotomy and organizing patient logistics.